Selective serotonin reuptake inhibitor attenuates the hyperresponsiveness of TLR2+ and TLR4+ Th17/Tc17-like cells in multiple sclerosis patients with major depression

Abstract

Elevated frequency of Th17-like cells expressing Toll-like receptors (TLRs) has been recently associated with relapsing-remitting multiple sclerosis (MS) pathogenesis, a chronic inflammatory demyelinating autoimmune disease of the central nervous system. We aimed to investigate the impact of current major depressive disorder (MDD) on the behaviour of these cells following in vitro stimulation with TLR2, TLR4, TLR5 and TLR9 agonists. Here, the level of both cell proliferation and cytokine production related to Th17/Tc17 phenotypes in response to TLR2 (Pam3C) and TLR4 (LPS) ligands was significantly higher in CD4⁺ and CD8⁺ T-cell cultures from MS/MDD patients when compared to non-depressed patients. These cytokine levels were positively associated with neurological disabilities in patients. No difference for responsiveness to TLR5 (flagellin) and TLR9 (ODN) agonists was observed. LPS, but not Pam3C, induced significant IL-10 release, mainly in patients without MDD. Interestingly, more intense expression of TLR2 and TLR4 on these cells was observed in MDD patients. Finally, in vitro addition of serotonin and treatment of MDD patients with selective serotonin reuptake inhibitors (SSRIs) reduced the production of Th17/Tc17-related cytokines by CD4⁺ and CD8⁺ T cells in response to Pam3C and LPS. However, only SSRI therapy diminished the frequency and intensity of TLR2 and TLR4 expression on circulating CD4⁺ and CD8⁺ T cells. In summary, although preliminary, our findings suggest that adverse events that elevate circulating levels of TLR2 and TLR4 ligands can affect MS pathogenesis, particularly among depressed patients.

Keywords: Multiple sclerosis; PAMP; TLR; Tc17; Th17; cytokines; major depressive disorder; selective serotonin reuptake inhibitors; serotonin.

Figure 1

Impact of MDD on circulating levels of cytokines in patients with MS and their relationship with the degree of neurological disability. In (A), plasma of MS patients with (n = 25) or without (n = 25) MDD was submitted to ELISA for quantification of IL‐1, IL‐6, TNF‐α, IFN, IL‐17 and IL‐10 cytokines. Data are shown as mean ±SD of seven independent experiments with 3 to 4 MS and 3 to 4 MS/MDD samples per experiment. Significance was calculated by comparing MS versus MS/MDD, and thepvalues are indicated in the figure. In (B), the correlation between cytokine levels and the EDSS score at the time of blood collection is shown

Figure 2

Comparative analysis of cytokine production by PBMC of patients with MS with or without MDD in response to different TLR ligands. The cytokine content in the supernatants collected from PBMC cultures (1 × 10⁶/ml) of MS patients with (MS/MDD,n = 25) or without (MS,n = 25) MDD was maintained for 2 days in the absence or presence of lipopolysaccharide (LPS, 100 gg/ml), Pam3Csk4 (Pam3C, 1 lg/ml), flagellin (FLA, 1 µg/ml) and oligodeoxynucleotide (ODN, 1 µM/ml). The cytokine concentrations [(A) IL‐1β, (B) IL‐6, (C) IL‐18, (D) IL‐18, (E) TNF‐α, (F) GM‐CSF, (G) IL‐12, (H) IFN‐γ, (I) IL‐23, (J) IL‐17, (K) IL‐21, (L) IL‐22 and (M) IL‐10] were determined using Luminex. Data are shown as mean ±SD of seven independent experiments with 3 to 4 MS and 3 to 4 MS/MDD samples per experiment. Significance was calculated by comparing MS versus MS/MDD, and (*), (**) and (***) indicatepvalues <0.05, <0.001 and <0.0001

Figure 3

Modulatory effects of serotonin (5‐HT) on cytokine production by PBMC of patients with MS with or without MDD in response to TLR2 and TLR4 ligands. The PBMC cultures (1 × 10⁶/ml) from MS patients with (MS/MDD,n = 25) or without (MS,n = 25) MDD were activated for 2 days with TLR2 (Pam3C, 1 µg/ml) and TLR4 (LPS, 100 ng/ml). In some cultures, 5‐HT (200 ng/ml) was added and the cytokine production [(A) IL‐1β, (B) IL‐6, (C) IL‐18, (D) IL‐18, (E) TNF‐α, (F) GM‐CSF, (G) IL‐23, (H) IL‐17, (I) IL‐22 and (J) IL‐10] assayed by Luminex. Data are shown as mean ±SD of seven independent experiments with 3 to 4 MS and 3 to 4 MS/MDD samples per experiment. Significance was calculated by comparing MS versus MS/MDD, and (*), (**) and (***) indicatepvalues <0.05, <0.001 and <0.0001

Figure 4

Comparison of the proportion of circulating IL‐17‐secreting CD4⁺and CD8⁺T cells expressing TLR2 and TLR4 in MS patients with or without MDD. The mean proportion of CD4⁺and CD8⁺T cells positive for TLR2 (B) and TLR4 (E), as well as MFI of TLR2 (C) and TLR4 (F) for these cells, was determined by cytometry following representative dot plots and histograms shown in panel A after acquisition of 200,000 events in samples obtained from healthy subjects (HS,n = 25) and MS patients with (MS/MDD,n = 25) or without (MS,n = 25) MDD. The mean frequency of IL‐17‐secreting CD4⁺and CD8⁺T cells among TLR2⁺(D) and TLR4⁺(G) cells was also evaluated after activation of those cells with PMA plus ionomycin. Data are shown as mean ±SD of seven independent experiments with 3 to 4 samples per experiment from each group (HS, MS and MS/MDD) (Figure2S). Significance was calculated by comparing different cell culture conditions from HS, MS and MS/MDD

Figure 5

Impact of MDD on proliferative response and cytokine production by CD4⁺and CD8⁺T cells from MS patients in response to different TLRs. Purified CD4⁺and CD8⁺T cells (0.5 × 10⁶/ml), obtained from MS patients with (MS/MDD,n = 15) or without (MS,n = 15) MDD, were maintained for 2 days in the presence of medium (none), lipopolysaccharide (LPS, 100 gg/ml), Pam3Csk4 (Pam3C, 1 lg/ml), flagellin (FLA, 1 µg/ml) or oligodeoxynucleotide (ODN, 1 µM/ml). In (A), the cell proliferation was determined by [³H^]] TdR uptake. The cytokine contents in the supernatants from (B) CD4⁺and (C) CD8⁺T‐cell cultures were evaluated by Luminex. Data are shown as mean ±SD of seven independent experiments with 2 to 3 samples per experiment from MS and MS/MDD patients (FigureS3). Significance was calculated, and thepvalues are shown in the figure

Figure 6

Effect of serotonin (5‐HT) on IL‐6, IL‐17 and IL‐10 produced by CD4⁺and CD8⁺T cells from MS and MS/MDD patients in response to LPS and Pam3C. Purified (A) CD4⁺and (B) CD8⁺T cells (0.5 × 10⁶/ml), obtained from MS [n =10 (O)] and MS/MDD [n =10 (•)] patients, were maintained for 2 days in the presence LPS (100 ng/ml) or Pam3Csk4 (Pam3C, 1 µg/ml). The cytokine levels were evaluated by Luminex. Data are shown as mean ±SD of five independent experiments with 4 samples per experiment (2 MS and 2 MS/MDD patients). Significance was calculated by comparing different cell culture conditions from MS versus MS/MDD patients, and thepvalues are shown in the figure

Figure 7

Impact of MDD treatment on TLR2 and TLR4 expression and cytokine release by CD4⁺and CD8⁺T cells from MS patients. Purified CD4⁺and CD8⁺T cells (0.5 × 10⁶/ml), obtained from 10 MS/MDD patients just before (t0) and 6 months (t1) after treatment with SSRIs, were analysed for TRL2 and TLR4 expression by cytometry following the strategy shown in the panel A. In (B) and (D), the mean frequency of CD4⁺and CD8⁺T cells positive for TLR2 and TLR4, respectively, is shown. In (C) and (E), the intensity of TLR2 and TLR4 expression on CD4⁺and CD8⁺T cells, respectively, is shown. In (F) and (G), the cytokines released by (F) CD4⁺(G) and CD8⁺T cells after stimulation by Pam3C (1 µg/ml) and LPS (100 ng/ml) for 2 days are presented. Data are shown as mean ±SD of five independent experiments with 4 samples per experiment (2 MS and 2 MS/MDD patients). Significance was calculated by comparing different cell culture conditions from MS versus MS/MDD, and thepvalues are shown in the figure